Identification ofXylella fastidiosaAntivirulence Genes: Hemagglutinin Adhesins Contribute toX. fastidiosaBiofilm Maturation and Colonization and Attenuate Virulence

Abstract

Xylella fastidosa, a gram-negative, xylem-limited bacterium, is the causal agent of several economically important plant diseases, including Pierce's disease (PD) and citrus variegated chlorosis (CVC). Until recently, the inability to transform or produce transposon mutants of X. fastidosa had been a major impediment to identifying X. fastidosa genes that mediate pathogen and plant interactions. A random transposon (Tn5) library of X. fastidosa was constructed and screened for mutants showing more severe symptoms and earlier grapevine death (hypervirulence) than did vines infected with the wild type. Seven hypervirulent mutants identified in this screen moved faster and reached higher populations than the wild type in grapevines. These results suggest that X. fastidosa attenuates its virulence in planta and that movement is important in X. fastidosa virulence. The mutated genes were sequenced and none had been described previously as antivirulence genes, although six of them showed similarity with genes of known functions in other organisms. One transposon insertion inactivated a hemagglutinin adhesin gene (PD2118), which we named HxfA. Another mutant in a second putative X. fastidosa hemagglutinin gene, PD1792 (HxfB), was constructed, and further characterization of these hxf mutants suggests that X. fastidosa hemagglutinins mediate contact between X. fastidosa cells, which results in colony formation and biofilm maturation within the xylem vessels.

Quorum-sensing regulation governs bacterial adhesion, biofilm development, and host colonization in Pantoea stewartii subspecies stewartii

“…Therefore, we suggest a functional corollary between bacterial biofilm development and xylem colonization similar to that described for Xylella fastidiosa infections of grape vine and Clavibacter michiganensis subsp. sepedonicus of potato (42)(43)(44)(45)(46)(47). Most significantly, our microscopic studies show that P. stewartii colonizes the xylem of corn with spatial specificity rather than by indiscriminate growth to fill the lumen of the xylem.…”

Genomic Insights into Xylella fastidiosa Interactions with Plant and Insect Hosts

“…Initial results of studies of virulence factors in X. fastidiosa involving the screening of random insertional mutants revealed a surprising role for adhesins. Specifically, hxfA and hxfB mutants that no longer expressed these two related hemagglutinin-like proteins exhibited a hyper-virulent phenotype in grape (Guilhabert and Kirkpatrick 2005). Furthermore, these mutants exhibited reduced cell-cell aggregation and moved further in grape xylem vessels in the wild-type strain after inoculation (Guilhabert and Kirkpatrick 2005).…”

“…Specifically, hxfA and hxfB mutants that no longer expressed these two related hemagglutinin-like proteins exhibited a hyper-virulent phenotype in grape (Guilhabert and Kirkpatrick 2005). Furthermore, these mutants exhibited reduced cell-cell aggregation and moved further in grape xylem vessels in the wild-type strain after inoculation (Guilhabert and Kirkpatrick 2005). Because such proteins would be expected to facilitate both cell-cell aggregation as well as cell surface attachment, it would be expected that their presence would tend to reduce the virulence of X. fastidiosa by impeding its movement along xylem vessels.…”

“…Although adhesion is essential for retention in insects, it limits colonization of plants compared to the rpfF mutant, highlighting the distinct requirements for life in such different hosts (Newman et al 2004;Guilhabert and Kirkpatrick 2005;Chatterjee et al 2008c). Thus, X. fastidiosa's conflicting life history is framed by the contrasting requirement to move within plants to increase its population size and thus its chances of being acquired by insects, but at the same time, it must increase its adhesiveness so it can attach to insects, which consequently reduces within-plant movement.…”

Characterization of Xylella fastidiosa popP Gene Required for Pathogenicitya

“…Pierce's disease symptoms were observed two months post inoculation. The symptoms were rated on a scale from 0 to 5 as described previously [28] with 0 representing healthy grapevines without any scorched leaves (water control) and 5 representing plants with severely scorching symptoms. The final disease index was an average from 5 independent replications in each X. fastidiosa strain.…”